Antibodies, through their exquisite ability to specifically target a distinct antigen on an endogenous cell, bacteria, virus, or toxin, constitute powerful therapeutic agents characterized by limited side effects. Several antibodies introduced onto the market over the past few years have achieved astonishing success in treating a variety of diseases, including cancer and inflammatory, cardiovascular, respiratory, and infectious diseases. There are currently approximately 480 launched and developmental antibody programs worldwide, 83% of which are located in the United States. Over 20% of all biopharmaceuticals currently being evaluated in clinical trials are antibodies, according to the Pharmaceutical Research Institute of America reports. The projected United States antibody market is anticipated to increase about ten-fold over the next decade, to $10.1 billion in 2010 (The Genesis Report: 25+ Business Development & Innovation Opportunities in Monoclonal Antibodies-Emerging Opportunities in 2010, The Genesis Group, Montclair, N.J.). In contrast to such efforts in antibody development, techniques for their purification, stabilization or subsequent delivery are often limited.
It is imperative that the higher order three-dimensional architecture or tertiary structure of an antibody be preserved until such time that the individual antibody molecules are required to perform their unique function. To date, a limiting factor for the use of antibodies, particularly in therapeutic regimens, remains the sensitivity of antibody structure to chemical and physical denaturation encountered during delivery. Various approaches have been employed to overcome these barriers. However, these approaches often incur loss of protein activity or the additional expense of protein stabilizing carriers or formulations.
The stability of small molecule crystalline drugs is such that they can withstand extreme forces during the manufacturing process (see U.S. Pat. No. 5,510,118). Such forces are associated with milling nanoparticles of crystalline material of relatively insoluble drugs and include: shear stress, turbulent flow, high impact collisions, cavitation and grinding. Small molecular crystalline compounds have been recognized as being much more stable toward chemical degradation than the corresponding amorphous solid [Pical, M. J., Lukes, A. L., Lang, J. E. and Gaines, J. Pharm. Sci. 67:767 (1978)].
To date, those of skill in the art recognize that the greatly enhanced stability of the crystalline state observed for small molecules does not translate to biological macromolecules, such as whole antibodies [Pical, M. J. and Rigsbee, D. R., Pharm. Res. 14:1379 (1997)]. For example, aqueous suspensions of crystalline insulin are only slightly more stable (to the degree of a factor of two) than corresponding suspensions of amorphous phase [Brange, J., Langkjaer, L., Havelund, S. and Volund, A., Pharm. Res. 9:715 (1992)]. In the solid state, lyophilized amorphous insulin is more stable than lyophilized crystalline insulin under all conditions investigated so far [Pical, M. J. and Rigsbee, D. R., Pharm. Res. 14:1379 (1997)]. However, using two model proteins, glucose oxidase and lipase, Shenoy et al. demonstrated that dry crystalline formulations can be significantly more stable than their amorphous counterparts [Shenoy, B. et al., Biotechnol Bioeng. 73(5):358-69 (2001)]. Surprisingly, the present invention provides crystals of whole antibodies and crystals of single-chain Fv (scFv) antibody fragments or Fab antibody fragments (the “ab” stands for “antigen-binding”) that are more stable than their soluble antibody or antibody fragment counterparts.
Despite recent progress in protein technology generally, two problems continue to limit the use of biological macromolecules in industry and medicine. The first problem relates to molecular stability and sensitivity of higher order tertiary structures to chemical and physical denaturation during manufacturing and storage. Second, the field of biological delivery of therapeutic proteins requires that vehicles be provided which release native proteins, such as whole antibodies, at a rate that is consistent with the needs of the particular patient or disease process.
Although crystallization of whole antibodies has been a subject of significant interest for the last three decades, very few whole antibodies have ever been crystallized and, even then, solely in the context of structural studies [Harris L. J., Skaletsky, E., and McPherson, A., J. Mol. Biol. 275:861-72 (1998); Harris L. J., Larson, S. B., Skaletsky, E., and McPherson, A., Immunological Reviews 163:35-43 (1998)]. All of these crystals were obtained by vapor diffusion techniques, which yielded only a very small quantity of crystals for structural analysis. Such yields were far below those required for pharmaceutical, diagnostic or other commercial applications. Furthermore, such low yields were largely attributed to the difficulties in antibody crystallization due to their relatively large size, the presence of oligosaccharides on their surfaces, and the high degree of their segmental flexibility.
Fab antibody fragments have also been crystallized, but solely for use in X-ray crystallographic structural studies [See, e.g., Ito et al., Acta Crystallogr. D. Biol. Crystalloqr. 57:1700-02 (2001); Covaceuszach et al., Acta Crystalloqr. D. Biol. Crystalloqr. 57:1307-09 (2001); Saul et al., Bioorg. Khim. 25:247-52 (1999); Pichla et al., J. Struct. Biol. 119:6-16 (1997); Maninder et al., J. Mol. Biol. 242:706-08 (1994)].
The following table provides a general comparison between crystallization for X-ray crystallographic structural studies and large-scale crystallization according to this invention:
X-RAYCRYSTALLOGRAPHICLARGE-SCALEPARAMETERSTUDIESCRYSTALLIZATIONCrystal size>500 μm0.1-100 μm(longestdimension)Crystal qualityVery importantLess importantGrowth rateNot importantImportantYieldNot importantVery importantPrecipitateUsually presentRarely present
Crystallization of whole antibodies, or fragments thereof, on a large scale, a process allowing an alternative route of delivery for therapeutic antibodies, has never before been explored.
Antibodies, and fragments thereof, are increasingly employed in the pharmaceutical, diagnostic and research industries. There is a great need for alternative stabilization procedures, which are fast, inexpensive and Moreover, stabilization procedures are needed that do not involve the excessive use of excipients, which can interfere with the functions of whole antibodies.
The present invention seeks to overcome barriers to the widespread use of antibodies for therapeutic and other biomedical purposes by providing methods for crystallizing whole antibodies, and fragments thereof, on a large scale.